We had a chance to share our plans with Prof. Jan van der Meer (UNIL) and Dr. Sabrina Leuenberger (Biosafety expert University of Bern) on the 27 March.
We decided to put together a presentation / demo of early prototype ideas per small group, so people could see and touch as well as hear what we planned to do.
Here is the summary of the feedback we received, along with the presentation pdfs.
Influence of lenses and filter (Marie W.) [pdf]:
The aim was to analyze the influence of lenses and filter on loss of fluorescence by looking at the spectrum of the light at the point of the detector.
In this way, we can hopefully see the difference between incident and fluorescent light, and understand what happens whether the signal gets absorbed in the plastic lens, and whether we can improve the signal to noise ratio with or without the filter.
For that the prototype will be modified in order to the lenses and the filter can be removed anytime and stably fix the spectruinio (DVD based spectrometer) instead of the sensor.
The received feedback was to include analysis on scattering.
Scattering and cell lysis (Dounia and Marie W.) :
We are going to compare the spectrum of different samples like dextran, beads, eGFP transformed e.coli and free eGFP protein obtained by cell lysis.
The first one will be the negative control because fluorescent dextran is fluorescent (similar wavelengths as eGFP), but will not scatter light.
Beads will be the positive control for scattering simulate the shape of the bacteria.
We will use non-fluorescent E.Coli to determine the light scattered, eGFP E. Coli for the fluorescence + scattering, and after the feedback, free eGFP to know how much light is scattered comparing to E.Coli cell.
We will try extracting eGFP from the bacteria using t-butanol with the Three-Phase Partitioning (TPP) method.
The sonification of Arsenic (Colombine and Marie D.) [pdf]
The aim of this part of the project is to improve the actual prototype to make it more intuitive and friendly for the users.
Instead of counts, which shows up as a number on an LCD screen, we would like to create a specific sound depending on the arsenic water concentration. Until now we managed to produce different sounds depending on the light frequency on a bread board. We need to adapt it to use music files, and then to adapt it to the prototype. We will finally create a script to test any real data.
The idea of making the prototype more accessible by turning the visual output into a sound output was a hit. Prof. Van der Meer even suggested that we could get rid of the problem caused by fluorescence and detect directly the sound instead by using bacteria that produce an electrical signal. This is an interesting idea to keep in mind. For the time being, we will test it on the current prototype with fluorescent dextrans mimicking the bacteria producing eGFP.
Arsenic detection with the LSS mOrange bioreporter (Mathilde) [pdf]:
The aim of this subject is to characterize a new fluorescence bioreporter, especially the strain, the sensitivity to arsenic, the time for development, the fluorescence activity… The first step of this part was to get familiar with the protocol and to compare the two reporters : eGFP and LSS mOrange. The following experiments could be :
- Make a precise curve of the relative fluorescence of LSS mOrange when arsenic concentration varies.
- Find an ideal buffer for LSS mOrange and AsrR synthesis.
- Compare the time that LSS mOrange and eGFP need to take their active conformations.
The feedback about the two first experiments is that their are interesting for the characterization of LSS mOrange, but for the third we have to ask if it is very essential to have a protein which get its active conformation very fast. Indeed, based on the paper : Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature methods, 2(12), 905-909, LSS mOrange has a half-time of maturation of 2.5 hours, which is quite long. But if we consider the time we take to test many samples and the fact that we don’t need immediately the result of the arsenic test, we can have a molecule which reacts after 5 hours.
Double cap for water sample collection (Mathilde) :
The motivations of making a double cap are :
- Assure secure samples collection in the field.
- Keep the GMO reporter bacteria from being released into the environment.
- Replace the syringe (which can be dangerous and produces extra wastes).
The prototype was well-received. The principal concern is about the sealing of the cap, but we will first prototype by 3D printing the double cap as a single piece. We also plan to return to BAFU – Federal Office for the Environment, and obtain their feedback.
There were also good ideas to improve the double cap, for example a system of clips which maintain the two caps together without any aluminum seal.